Polyketides and non-ribosomal peptides are in a class of natural products important both as drug sources and as dangerous food toxins and virulence factors. While studies over the last two decades have provided substantial characterization of the modular synthases that produce these compounds at the genetic level, their understanding at the protein level is much less understood. Our laboratory has recently developed a proteomic platform, called an orthogonal active site identification system (OASIS), for identifying and quantifying natural product synthase enzymes. OASIS leverages activity based probes for enzyme enrichment in conjunction with highly sensitive peptide sequencing. To date, we have demonstrated the value of OASIS for the proteomic comparison of two model strains of Bacillus brevis. In this program, we apply this new tool to address topical problems in the discovery and study of modular synthases. Within this aim we have identified case studies that examine: antitubercular natural products from select sphagnum bog bacterial strains; the anticancer polyketide spirohexenolide B from Streptomyces platensis; the toxic virulence factor mycolactone from Mycobacterium ulcerans; and the shellfish toxin okadaic acid from the marine dinoflagellate Prorocentrum lima. Each of these studies offers solutions to important biomedical problems that have yet to be determined and remain currently unstudied due to the limitations of genetic techniques. The application of OASIS offers a new opportunity to evaluate these pathways through combining specific synthase-selective probes with proteomic technology, opening the tools of contemporary proteomics to natural product sciences. This program aims to bring the current state of natural product science into the proteomic era through the optimization and application of an orthogonal active site identification system (OASIS). The OASIS system provides new avenues into the study of natural product biosynthesis by enabling novel connectivity with mass spectral based proteomic analyses. Applications of the technique are examined within the context of natural product discovery, disease research, and the evaluation of dangerous toxins and virulence factors. PUBLIC HEALTH RELEVANCE: This program aims to bring the current state of natural product science into the proteomic era through the optimization and application of an orthogonal active site identification system (OASIS). The OASIS system provides new avenues into the study of natural product biosynthesis by enabling novel connectivity with mass spectral based proteomic analyses. Applications of the technique are examined within the context of natural product discovery, disease research, and the evaluation of dangerous toxins and virulence factors.